Monoclonal antibodies to tacrolimus and immunoassay methods for tacrolimus

ABSTRACT

An IgG 1  λ monoclonal antibody to the immunosuppressive drug tacrolimus has improved properties. In particular, this monoclonal antibody, designated 1H6, has reduced cross-reactivity to several tacrolimus metabolites. This antibody is suitable for performance of immunoassays such as homogeneous immunoassays to detect or determine the presence or concentration of tacrolimus in samples such as blood samples. The invention further includes derivatives of tacrolimus derivatized at a non-binding portion of the molecule useful in immunizing antibody-producing animals and in producing such monoclonal antibodies, as well as labeled derivatives of tacrolimus useful as tacrolimus analogues in such assays. The invention further includes immunoassay methods for the detection of tacrolimus and test kits useful in performing such immunoassays.

FIELD OF THE INVENTION

This invention is directed to monoclonal antibodies to tacrolimus,methods for producing such monoclonal antibodies, and derivatives oftacrolimus useful for producing such monoclonal antibodies.

BACKGROUND OF THE INVENTION

Tacrolimus is a macrolide isolated from Streptomyces tsukubaensis.(butt) Tacrolimus has the chemical name[3S-[3R*[E(1S*,3S*,4S*)],4S*,5R*,8S*,9E,12R*,14R*,15S*,16R*,18S*,19S*,26aR*]]-5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro-5,19-dihydroxy-3-[2-(4-hydroxy-3-methoxycylohexyl)-1-methylethenyl]-14,16-dimethoxy-4,10,12,18-tetramethyl-8-(2-propenyl)-15,19-epoxy-3H-pyrido[2,1-c][1,4]-oxaazacyclotricosine-1,7,20,21(4H, 23H) tetrone. The structure of tacrolimus, giving the numbering, isshown below in FIG. 16.

Tacrolimus is also known as FR-900506 or FK-506. Tacrolimus hasimmunosuppressive activity and antimicrobial activity.

The immunosuppressive activity of tacrolimus is particularly importantand has led to the increasingly wide use of this drug. Immunosuppressionis used clinically in a number of contexts, most importantly inpreventing rejection in organ transplantation. Immunosuppressive drugsare also administered in prevention of Rh hemolytic disease of thenewborn and in the treatment of autoimmune disorders. Tacrolimusinhibits T-cell activation by binding to a cytosolic protein known asFKBP (FK506 binding protein). The drug-binding protein complex stablyassociates with calcineurin. This inhibits the serine-threoninephosphatase activity of this Ca²⁺-dependent enzyme. This inhibitscalcineurin-dependent activation of lymphokine expression, apopotosis,and degranulation (G. Wiederrecht et al., “The Mechanism of Action ofFK-506 and Cyclosporin A,” Ann. N.Y. Acad. Sci. 696:9-19 (1993)).

Tacrolimus can be administered intravenously in a short or continuousinfusion or orally. Tacrolimus, like other immunosuppressant agents, hasa spectrum of toxicity. The major toxicity associated with clinical useof the drug is nephrotoxicity. In addition, neurotoxicity can develop,associated with headache, tremor, insomnia, pain, or other symptoms.Additionally, gastrointestinal toxicity manifested by diarrhea or nauseacan develop, as can cardiovascular toxicity manifested by hypertension.

Additionally, metabolic toxicity can develop as manifested by thedevelopment of such symptoms as hyperkalemia, hypomagnesemia, orhyperglycemia. In addition, long-term immunosuppression with tacrolimuscan produce increased risk of all types of infections, not only theusual bacterial, viral, and fungal pathogens, but also various unusualopportunistic infections as well.

Additionally, there is an increased risk of lymphomas and relatedmalignancies associated with the administration of tacrolimus (M. L.Cleary & J. Sklar, “Lymphoproliferative Disorders in Cardiac TransplantRecipients are Multiclonal Lymphomas,” Lancet 2:49-493 (1984); L. J.Swinnen et al., “Increased Incidence of Lymphoproliferative DisorderAfter Immunosuppression with a Monoclonal Antibody OKT3 inCardiac-Transplant Recipients,” N. Engl. J. Med. 323:1723-1728 (1990)).At least some of these malignancies are related to impaired immuneresponses to Epstein-Barr virus (B. Z. Katz et al., “Latent andReplicating Forms of Epstein-Barr virus DNA and Lymphomas inLymphoproliferative Diseases,” J. Infect. Dis., 160:589-598 (1989)).

The potency and the spectrum of toxicities of tacrolimus requiressensitive, reproducible, and reliable methods for monitoring the bloodconcentration of these compounds after administration to a patient, sucha patient undergoing organ transplantation. It is important that suchmethods be sensitive enough to detect low concentrations of tacrolimus.It is also important that such methods be reliable and reproducible, andavoid interference from compounds such as metabolites of tacrolimus.

Although antibodies and immunoassays to tacrolimus exist, and aredescribed, for example, in U.S. Pat. No. 5,532,137 to Niwa et al.,incorporated herein by this reference, there is still a need for thedevelopment of improved antibodies and immunoassays specific fortacrolimus. There is, particularly, a need for improved monoclonalantibodies to tacrolimus that can be used in developing a sensitive,reliable, and reproducible immunoassay for tacrolimus.

The development of a reliable immunoassay for tacrolimus is complicatedby the fact that tacrolimus has a number of metabolites that are foundin the blood of an individual being treated with tacrolimus. Theconversion of tacrolimus to these metabolites involve demethylation,hydroxylation, and ring formation. It is important that antibodies totacrolimus have as little cross-reactivity with these derivatives aspossible.

There is therefore a need for the development of improved monoclonalantibodies to tacrolimus that are useful for immunoassays for tacrolimusand that possess a minimal degree of cross-reactivity to tacrolimusmetabolites. There is also a need for improved immunoassays using suchmonoclonal antibodies for the detection and determination of tacrolimusin the blood of patients being administered tacrolimus.

SUMMARY

We have discovered that tacrolimus, when derivatized in the non-bindingdomain, can be coupled to a high molecular weight carrier such as aprotein for immunization to produce antibodies. The resultingantibody-producing cells can be used to generate monoclonal antibodiesby cell fusion. These monoclonal antibodies have desirable properties,including reduced cross-reactivity with tacrolimus metabolites.

One embodiment of the present invention is a monoclonal antibody totacrolimus that is a monoclonal antibody designated 1H6 and which hasless than about 8% cross-reactivity with each of 15-demethyl tacrolimus,31-demethyl tacrolimus, 13,31-didemethyl tacrolimus, 15,31-didemethyltacrolimus and 12-hydroxy tacrolimus.

Another embodiment of the present invention is a monoclonal antibody totacrolimus that:

(1) competes with the IgG₁λ monoclonal antibody designated 1H6 at leastabout 80% as effectively on a molar basis as compared with the IgG₁λmonoclonal antibody designated 1H6 as measured by competition assays;and

(2) has less than about 10% cross-reactivity with each of 15-demethyltacrolimus, 31-demethyl tacrolimus, 13,31-didemethyl tacrolimus,15,31-didemethyl tacrolimus, and 12-hydroxy tacrolimus. Preferably, themonoclonal antibody to tacrolimus competes at least about 90% aseffectively on a molar basis as the monoclonal antibody designated 1H6and has less than about 8% cross-reactivity with each of 15-demethyltacrolimus, 31-demethyl tacrolimus, 13,31-didemethyl tacrolimus,15,31-didemethyl tacrolimus and 12-hydroxy tacrolimus.

Another embodiment of the present invention is a hybridoma producing theIgG₁λ monoclonal antibody to tacrolimus designated 1H6 as describedabove.

Yet another embodiment of the present invention is a hybridoma producinga monoclonal antibody that:

(1) competes with the IgG₁λ monoclonal antibody designated 1H6 at leastabout 80% as effectively on a molar basis as compared with the IgG₁λmonoclonal antibody designated 1H6 as measured by competition assays;and

(2) has less than about 10% cross-reactivity with each of 15-demethyltacrolimus, 31-demethyl tacrolimus, 13,31-didemethyl tacrolimus,15,31-didemethyl tacrolimus, and 12-hydroxy tacrolimus.

Another embodiment of the present invention is a monoclonal antibodythat:

(1) competes with the IgG₁λ monoclonal antibody designated 1H6 at leastabout 80% as effectively on a molar basis as compared with the IgG₁λmonoclonal antibody designated 1H6 as measured by competition assays;and

(2) has less than about 10% cross-reactivity with each of 15-demethyltacrolimus, 31-demethyl tacrolimus, 13,31-didemethyl tacrolimus,15,31-didemethyl tacrolimus, and 12-hydroxy tacrolimus, wherein at leastsome of the constant regions of the antibody are replaced by humanconstant regions so that the monoclonal antibody is humanized.

Yet another embodiment of the present invention is a single-chainrecombinant antibody (sFv) including therein the variable regions of anantibody to tacrolimus that:

(1) competes with the IgG₁λ monoclonal antibody designated 1H6 at leastabout 80% as effectively on a molar basis as compared with the IgG₁λmonoclonal antibody designated 1H6 as measured by competition assays;and

(2) has less than about 10% cross-reactivity with each of 15-demethyltacrolimus, 31-demethyl tacrolimus, 13,31-didemethyl tacrolimus,15,31-didemethyl tacrolimus, and 12-hydroxy tacrolimus.

Still another embodiment of the present invention is a monoclonalantibody to tacrolimus produced by fusion of antibody-producing cellsfrom an antibody-producing mammal immunized with tacrolimus derivatizedwith a carboxymethyl oxime moiety at a carbon atom in the non-bindingdomain of tacrolimus conjugated to a high molecular weight protein witha suitable fusion partner. Preferably, the carbon atom in thenon-binding domain of tacrolimus is carbon 22. Preferably, the highmolecular weight protein is keyhole limpet hemocyanin.

Yet another embodiment of the present invention is a monoclonal antibodyto tacrolimus that is an IgG₁λ monoclonal antibody, that has a bindingaffinity for tacrolimus of about 3.7×10⁹ liters/mole, that cross-reactswith 13-demethyl tacrolimus, and that has less than about 8%cross-reactivity to all of the following tacrolimus metabolites:15-demethyl tacrolimus; 31-demethyl tacrolimus; 13,31-didemethyltacrolimus; 15,31-didemethyl tacrolimus; and 12-hydroxy tacrolimus.

Another aspect of the present invention is antibodies, includingpolyclonal antibodies, produced by immunization of an antibody-producingmammal with tacrolimus derivatized with a carboxymethyl oxime moiety ata carbon atom in the non-binding domain of tacrolimus conjugated to ahigh molecular weight protein. Preferably, the carbon atom in thenon-binding domain of tacrolimus is carbon 22. Preferably, the highmolecular weight protein is keyhole limpet hemocyanin.

Another aspect of the present invention is a conjugate comprising anantibody according to the present invention directly or indirectlyconjugated to a detectable label. The detectable label can be selectedfrom the group consisting of an enzyme label, a radioactive label, afluorescent label, a chemiluminescent label, a bioluminescent label, anda particulate label. In many applications, the label is preferably anenzyme label.

Still another aspect of the present invention is a method of detectingor determining tacrolimus comprising the steps of:

(1) providing a sample suspected of containing tacrolimus;

(2) reacting the sample with:

-   -   (a) an antibody according to the present invention; and    -   (b) optionally, a tacrolimus analogue; wherein one of the        antibody or the tacrolimus analogue is labeled with a label        producing a detectable signal; and

(3) observing or measuring one of:

-   -   (a) the signal associated with tacrolimus bound to antibody;    -   (b) the signal associated with tacrolimus unbound to antibody;        or    -   (c) the total signal present;        in order to detect or determine the presence or concentration of        tacrolimus in the sample.

Typically, in this method, the sample is reacted with a tacrolimusanalogue labeled with an enzyme label and the total signal present isobserved or measured to detect or determine the presence orconcentration of tacrolimus in the sample.

Another aspect of he present invention is a test kit comprising,packaged in separate containers:

(1) an antibody according to the present invention; and

(2) a tacrolimus analogue labeled directly or indirectly with an enzymelabel.

Yet another aspect of the present invention is a derivative oftacrolimus comprising tacrolimus that is derivatized with acarboxymethyl oxime moiety at a carbon atom in the non-binding domain oftacrolimus. Preferably, the carbon atom in the non-binding domain oftacrolimus is carbon 22.

Still another aspect of the present invention is a conjugate comprisinga derivative of tacrolimus as described above conjugated to a highmolecular weight protein. Preferably, the high molecular weight proteinis keyhole limpet hemocyanin.

Yet another aspect of the present invention is a method of derivatizingtacrolimus comprising reacting tacrolimus with carboxymethoxylamine toproduce a carboxymethyl oxime derivative of tacrolimus, thecarboxymethyl oxime moiety being located at carbon atom 22.

Still another aspect of the present invention is a method of producing aconjugate of tacrolimus with a high molecular weight protein comprising:

(1) reacting tacrolimus with carboxymethoxylamine to produce acarboxymethyl oxime derivative of tacrolimus, the carboxymethyl oximemoiety being located at carbon atom 22;

(2) activating the carboxymethyl oxime to produce a reactiveN-hydroxysuccinimide ester; and

(3) reacting the N-hydroxysuccinimide ester with the high molecularweight protein to produce the conjugate.

Another aspect of the present invention is a derivative of tacrolimuscomprising tacrolimus substituted with a carboxymethyl oxime moiety atcarbon atom 22 linked through a linker to a biotin moiety. In onepreferred embodiment of this aspect of the present invention, the linkerhas the structure NH₂—CH₂—CH₂—NH—CO—(CH₂)₅—NH₂, and one amine group ofthe linker forms an amide bond with the carboxyl group of thecarboxymethyl oxime and the other amine group of the linker forms anamide bond with the carboxyl group of the biotin.

Still another aspect of the present invention is a method ofderivatizing tacrolimus comprising:

(1) reacting tacrolimus with carboxymethoxylamine to produce acarboxymethyl oxime derivative of tacrolimus, the carboxymethyl oximederivative being located at position 22;

(2) activating the carboxymethyl oxime to produce a reactiveN-hydroxysuccinimide ester; and

(3) reacting the N-hydroxysuccinimide ester with the carboxyl group ofbiotin or a biotin derivative or analogue.

Yet another aspect of the present invention is a derivative oftacrolimus comprising tacrolimus that is derivatized with a bromoacetylmoiety at a carbon atom in the non-binding domain of tacrolimus.Preferably, the carbon atom in the non-binding domain is carbon atom 22.These derivatives can then be reacted with an enzyme or other protein toproduce a conjugate containing the derivative conjugated to a protein,such as an enzyme. In one preferred embodiment, the protein is acysteine-containing mutein of glucose-6-phosphate dehydrogenase.

Accordingly, another aspect of the present invention is a method ofderivatizing tacrolimus comprising:

(1) reacting tacrolimus with carboxymethoxylamine to produce acarboxymethyl oxime derivative of tacrolimus, the carboxymethyl oximederivative being located at position 22;

(2) activating the carboxymethyl oxime to produce a reactiveN-hydroxysuccinimide ester; and

(3) reacting the N-hydroxysuccinimide ester with the trifluoroaceticacid salt of bromoacetyl ethylenediamine to produce a bromoacetylderivative of tacrolimus.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with reference to the followingdescription, appended claims, and accompanying drawings where:

FIG. 1 is a mass spectrogram of the product resulting from the reactionof carboxymethoxylamine with the macrolide antibiotic FK-520, closelyrelated in structure to tacrolimus;

FIG. 2 is a portion of the mass spectrogram of FIG. 1, shown in expandedresolution centered around the predominant peak;

FIG. 3 is a mass spectrogram of the product resulting from the reactionof carboxymethoxylamine with tacrolimus;

FIG. 4 is a portion of the mass spectrogram of FIG. 3, shown in expandedresolution centered around the predominant peak;

FIG. 5 is a ¹H NMR spectrum at 250 MHz in CDCl₃ of the product resultingfrom the reaction of carboxymethoxylamine with tacrolimus;

FIG. 6 is a ¹³C NMR spectrum at 250 MHz in CDCl₃ of the productresulting from the reaction of carboxymethoxylamine with tacrolimus;

FIG. 7 is a ³C NMR spectrum at 500 MHz in CDCl₃ of tacrolimus shown as areference;

FIG. 8 is a mass spectrogram of the product resulting from the reactionof LC-biotin with tacrolimus monooxime;

FIG. 9 is a portion of the mass spectrogram of FIG. 8, shown in expandedresolution centered around the predominant peak

FIG. 10 is a ¹H NMR spectrum at 250 MHz in CDCl₃ of the productresulting from the reaction of LC-biotin with tacrolimus monooxime;

FIG. 11 is a calibration curve of a homogeneous enzyme immunoassay oftacrolimus using a monoclonal antibody produced by immunization of micewith a conjugate of tacrolimus derivatized at position 22 with acarboxymethyl oxime linked to keyhole limpet hemocyanin and cell fusionof the resulting antibody-producing cells with a fusion partner;

FIG. 12 is a graph showing the correlation between the results for theassay of tacrolimus using a homogeneous enzyme immunoassay with themonoclonal antibody for which the calibration curve is shown in FIG. 11and the results for the assay of tacrolimus using a method employing gaschromatography and tandem mass spectroscopy (LC/MS/MS);

FIG. 13 is a graph showing the correlation between the results for theassay of tacrolimus using the homogeneous enzyme immunoassay and usingLC/MS/MS on a panel of 70 patients with liver damage to whom tacrolimushad been administered;

FIG. 14 is a graph showing the correlation between the results for theassay of tacrolimus using the homogeneous enzyme immunoassay and using acommercially available immunoassay for tacrolimus for the panel of 70patients of FIG. 13;

FIG. 15 is a graph showing a Bland-Altman difference analysis plot forthe results of FIGS. 13-14; and

FIG. 16 is a drawing of the structural formula for tacrolimus, showingthe numbering of the molecule.

DESCRIPTION Definitions

As used herein, the terms defined below have the following meaningsunless otherwise indicated:

“Antibody”: As used herein the term “antibody” includes both intactantibody molecules of the appropriate specificity, and antibodyfragments (including Fab, F(ab′), Fv and F(ab′)₂), as well as chemicallymodified intact antibody molecules and antibody fragments, includinghybrid antibodies assembled by in vitro association of subunits. Alsoincluded are single-chain antibody molecules generally denoted by theterm sFv and humanized antibodies in which some or all of the originallynon-human constant regions are replaced with constant regions originallyderived from human antibody sequences. Both polyclonal and monoclonalantibodies are included unless otherwise specified; in a number ofcontexts, monoclonal antibodies are specifically specified. Additionallyincluded are modified antibodies or antibodies conjugated to labels orother molecules that do not block or alter the binding capacity of theantibody.

“Nucleic Acid Sequence”: The term “nucleic acid sequence” includes bothDNA and RNA unless otherwise specified, and, unless otherwise specified,includes both double-stranded and single-stranded nucleic acids. Alsoincluded are hybrids such as DNA-RNA hybrids. In particular, referenceto DNA includes RNA that has either the equivalent base sequence exceptfor the substitution of uracil in RNA for thymine in DNA, or has acomplementary base sequence except for the substitution of uracil forthymine, complementarity being determined according to the Watson-Crickbase pairing rules. Additionally, a reference to a nucleic acid sequenceincludes its complement according to the Watson-Crick base pairing rulesunless otherwise specified.

We have developed an improved monoclonal antibody to tacrolimus based onthe use of tacrolimus derivatized at a carbon atom within thenon-binding domain, preferably carbon-22. Polyclonal antibodies producedby immunization of antibody-producing animals with this immunogen arefirst made. The resulting antibody-producing cells are then used forcell fusion with a suitable fusion partner to produce hybridomas. Theresulting monoclonal antibodies produced by the hybridomas areparticularly useful in immunoassays for the detection of tacrolimus.

I. Derivatives of Tacrolimus

Accordingly, one aspect of the present invention is derivatives oftacrolimus derivatized at a carbon atom within the non-binding domain oftacrolimus. Preferably, the derivative is derivatized at the carbon-22position. One particularly preferred class of derivatives involvesreacting the keto group at the 22 position with an amine to produce anoxime. A particularly preferred amine is carboxymethoxylamine. Thereaction of tacrolimus with carboxymethoxylamine produces acarboxymethyl oxime.

This reaction involves reacting tacrolimus with carboxymethoxylamine inmethanol in the presence of sodium acetate to give the oxime.Accordingly, another aspect of the present invention is a method ofderivatizing tacrolimus comprising reacting tacrolimus withcarboxymethoxylamine to produce a carboxymethyl oxime derivative oftacrolimus, the oxime moiety being located at carbon-22. Further detailsof this reaction are given in Example 1.

Accordingly, another aspect of the present invention is a conjugatecomprising the derivative of tacrolimus which comprises tacrolimusderivatized with a carboxymethyl oxime moiety at position 22 conjugatedto a high molecular weight protein through the oxime moiety. Typically,the high molecular weight protein is a protein that is a suitablecarrier for haptens and can be, but is not necessarily limited to,proteins such as bovine serum albumin, thyroglobulin, ovalbumin,fibrinogen, or keyhole limpet hemocyanin. A particularly preferredcarrier is keyhole limpet hemocyanin. Alternatively, the high molecularweight protein can be an enzyme, such as an enzyme producing adetectable signal, such as glucose-6-phosphate dehydrogenase or alkalinephosphatase. Such conjugates are particularly useful in immunoassays fortacrolimus, as discussed below.

The preparation of these conjugates is set out in more detail below inExample 2. In general however, a method of preparation of the conjugateof tacrolimus with the high molecular weight protein is as follows: (1)preparation of the carboxymethyl oxime of tacrolimus as described above;(2) activating the carboxymethyl oxime to produce a reactiveN-hydroxysuccinimide ester; and (3) reacting the N-hydroxysuccinimideester with the high molecular weight protein to produce the conjugate.This reaction is discussed in detail in Example 2. The activation of thecarboxymethyl oxime to produce the N-hydroxysuccinimide ester typicallyis performed using a coupling agent such as a water-solublecarbodiimide. A preferred water-soluble carbodiimide is−3-(3-dimethylaminopropyl 1-ethyl-3-dimethylaminopropyl)-carbodiimidehydrochloride (EDAC). Other water-soluble carbodiimides are known in theart and can also be used.

In addition to forming conjugates with high molecular weight proteinssuch as keyhole limpet hemocyanin, the present invention alsoencompasses derivatives of tacrolimus substituted with a carboxymethyloxime moiety at carbon 22 linked through a linker to a biotin moiety.The linker can take one of a number of forms and can have differentlengths. The use of linkers between biotin and a hapten or antigen iswell known in the art and need not be described further in detail here.In one particularly preferred derivative, the linker has the structureNH₂—CH₂—CH₂—NH—CO—(CH₂)₅—NH₂. One of the amine groups of the linkerforms an amide bond with the carboxyl group of the carboxymethyl oxime,and the other amine group forms an amide bond with the carboxyl group ofthe biotin.

The length of this spacer can be changed by inserting or deleting one ormore CH₂ (methylene) groups at the two places in the spacer that have 2or more methylene groups.

The derivatives can be formed by reacting a N-hydroxysuccinimide esteras described above with the carboxyl group of biotin or a biotinderivative or analogue.

This reaction can be performed as described above.

In general, the method of forming such biotin derivatives comprises:

(1) reacting tacrolimus with carboxymethoxylamine to produce acarboxymethyl oxime derivative of tacrolimus, the carboxymethyl oximederivative being located at position 22;

(2) activating the carboxymethyl oxime to produce a reactiveN-hydroxysuccinimide ester; and

(3) reacting the N-hydroxysuccinimide ester with the carboxyl group ofbiotin or a biotin derivative or analogue.

In one preferred alternative, as discussed below in Example 3, the oximeis reacted with EDAC and N-hydroxysuccinimide in dimethylformamide(DMF). The activated product is then reacted with the biotin containingthe linker, such as LC-biotin.

Still another embodiment of conjugates of tacrolimus derivatized at acarbon atom within the non-binding domain of tacrolimus, preferablyposition 22, is a bromoacetyl derivative. The preparation of bromoacetylderivatives of tacrolimus derivatized at position 22 is described inExample 4. In general, the preparation of such derivatives comprises:

(1) reacting tacrolimus with carboxymethoxylamine to produce acarboxymethyl oxime derivative of tacrolimus, the carboxymethyl oximederivative being located at position 22;

(2) activating the carboxymethyl oxime to produce a reactiveN-hydroxysuccinimide ester; and

(3) reacting the N-hydroxysuccinimide ester with the trifluoroaceticacid salt of bromoacetyl ethylenediamine to produce a bromoacetylderivative.

The carboxymethyl oxime derivative used in this method is prepared asdescribed above.

Such bromoacetyl derivatives can be used to produce enzyme conjugates oftacrolimus by reacting the bromoacetyl moiety with a sulfhydryl group ofan enzyme, such as a mutein of glucose 6-phosphate dehydrogenase thatcontains a cysteine residue. The bromoacetyl derivative can react withother cysteine groups in other enzymes or proteins to produce otherenzyme or protein conjugates of tacrolimus.

II. Monoclonal and Polyclonal Antibodies and Hybridomas

Another aspect of the present invention is monoclonal antibodies andhybridomas producing them, as well as polyclonal antibodies produced byimmunization of antibody-producing animals with tacrolimus derivativesas described above.

One preferred embodiment of a monoclonal antibody to the presentinvention is a murine monoclonal antibody to tacrolimus that is a IgG₁λmonoclonal antibody designated 1H6. This monoclonal antibody reacts with13-demethyl tacrolimus but has less than about 8% cross-reactivity toall of the following tacrolimus metabolites: 15-demethyl tacrolimus;31-demethyl tacrolimus; 13,31-didemethyl tacrolimus; 15,31-didemethyltacrolimus; and 12-hydroxy tacrolimus. This monoclonal antibody has anestimated affinity for binding to tacrolimus of 3.7×10⁹ liters/mole.

Another monoclonal antibody according to the present invention is amonoclonal antibody to tacrolimus that: (1) competes with the IgG₁λmonoclonal antibody designated 1H6 at least about 80% as effectively ona molar basis as compared with the IgG₁λ monoclonal antibody designated1H6 as measured by competition assays; and (2) has less than about 10%cross reactivity with each of the following tacrolimus metabolites:15-demethyl tacrolimus; 31-demethyl tacrolimus; 13,31-didemethyltacrolimus; 15,31-didemethyl tacrolimus; and 12-hydroxy tacrolimus.

Preferably, the monoclonal antibody competes at least about 90% aseffectively on a molar basis as the monoclonal antibody designated 1H6and has less than about 8% cross reactivity with each of thesetacrolimus metabolites.

Another aspect of the present invention is a hybridoma producing amonoclonal antibody as described above. This includes a hybridomaproducing the IgG₁λ monoclonal antibody to tacrolimus designated as 1H6.This also includes a hybridoma producing a monoclonal antibody asdescribed above which competes at least about 80% as effectively on amolar basis with that antibody and has a limited degree ofcross-reactivity with tacrolimus metabolites as described above.

Another embodiment of the present invention is humanized monoclonalantibodies. In some applications it is preferred to have humanizedmonoclonal antibodies in which at least some of the constant regions ofthe antibody are replaced by human constant regions so that themonoclonal antibody is humanized. Typically, such procedures involve thegrafting of the murine complementarity-determining regions (CDRs) onto ahuman antibody. Additional alterations of individual amino acids withinthe framework may be necessary to recreate the antigen-binding site.Typically, this technique involves amplification of cDNA for the heavyand light variable chains of the murine hybridoma using the polymerasechain reaction (PCR) with a subset of synthetic oligonucleotide primerswith a small level of degeneracy. Mutagenesis, if required, can becarried out by standard methods. Typically, humanized V genes carryingthe variable regions are cloned into suitable expression vectors andexpressed in cells such as COS cells or CHO-K1 cells. Other mutationscan be made as needed. The preparation of such humanized monoclonalantibodies is generally known in the art and is described, for examplein C. A. K. Borrebaeck, “Antibody Engineering” 2d. ed., OxfordUniversity Press, New York, 1995), incorporated herein by thisreference.

Therefore, another embodiment of the present invention is a single-chainrecombinant antibody (sFv) including therein the variable regions of anantibody to tacrolimus that: (1) competes with the IgG₁λ monoclonalantibody designated 1H6 and described above at least about 80% aseffectively on a molar basis as compared with the IgG₁λ monoclonalantibody designated 1H6 as measured by competition assays; and (2) hasless than about 10% cross-reactivity with each of 15-demethyltacrolimus, 31-demethyl tacrolimus, 13,31-didemethyl tacrolimus,15,31-didemethyl tacrolimus, and 12-hydroxy tacrolimus.

Strategies for producing sFv single-chain antibodies are well known inthe art and are described, for example, in C. A. K. Borrebaeck,“Antibody Engineering,” supra, incorporated herein by this reference. Ingeneral, constructing an sFv involves manipulation of heavy- andlight-chain variable regions, which must be connected at the gene levelby an oligonucleotide sequence of codons for an appropriate peptidelinker. The linker must connect the V_(H) and V_(L) domains of thechosen Fv without perturbing interdomain contacts or interfering withdomain folding. A typically used linker is a 15-residue peptide thatconsists of 3 repeating units, each of which has four glycine residuesfollowed by a serine residue (J. S. Huston et al., “Protein Engineeringof Antibody Binding Sites: Recovery of Specific Activity in anAnti-Digoxin Single-Chain Fv Analog Produced in Escherichia coli,” Proc.Natl. Acad. Sci. USA 85:5879-5883 (1988)). Typically, such single-chainantibodies are produced in inclusion bodies in bacteria. Activitytypically requires refolding of the expressed polypeptides frominclusion bodies; conditions for performing this are generally wellknown.

Accordingly, such single-chain antibodies or sFv are also within thescope of the present invention.

Another embodiment of the present invention is a monoclonal antibody totacrolimus produced by fusion of antibody-producing cells from anantibody-producing mammal immunized with tacrolimus derivatized with acarboxymethyl oxime moiety at a carbon atom in the non-binding domain oftacrolimus conjugated to a high molecular weight protein with a suitablefusion partner. Preferably, the carbon atom in the non-binding domain oftacrolimus is carbon 22. As indicated above, typically, the highmolecular weight protein used for immunization is keyhole limpethemocyanin.

The preparation of monoclonal antibodies is well known in the art andneed not be described further here. For example, the production ofmonoclonal antibodies is described in J. W. Goding, “MonoclonalAntibodies: Principles and Practice” (2d. ed., Academic Press, London,1986), incorporated herein by this reference.

In general, the first step in the procedure is the production ofpolyclonal antibodies by standard techniques, such as immunization of anantibody-producing animal such as a mouse, a rat, a goat, a sheep, or acow with the antigen. The antigen is typically coupled to a highmolecular weight carrier such as a high molecular weight protein asdiscussed above. Immunization can be performed with or without anadjuvant such as complete Freund's adjuvant or other adjuvants such asmonophosphoryl lipid A and synthetic trehalose dicorynomycolateadjuvant; it is generally preferred to immunize with an adjuvant. Thenext step is to isolate spleen cells from antibody-producing animals andfuse the antibody-producing spleen cells with an appropriate fusionpartner, typically a myeloma cell, such as by the use of polyethyleneglycol or other techniques. Typically, the myeloma cells used are thosethat grow normally in hypoxanthine-thymidine (HT) medium but cannot growin hypoxanthine-aminopterin-thymidine (HAT) medium, used for selectionof the fused cells. The next step is selection of the fused cells,typically by selection in HAT medium. The next step is to screen thecloned hybrids for appropriate antibody production using immunoassayssuch as enzyme-linked immunosorbent assay (ELISA) or other immunoassaysappropriate for screening. Again, these steps are well known in the artand need not be described in further detail.

Another embodiment of the present invention is an antibody, which can bepolyclonal antibody, to tacrolimus produced by immunization of anantibody-producing mammal with tacrolimus derivatized with acarboxymethyl oxime moiety at a carbon atom in the non-binding domain oftacrolimus conjugated to a high molecular weight protein as describedabove. Preferably, the carbon atom in the non-binding domain oftacrolimus is carbon 22.

Another aspect of the present invention is a conjugate comprising anantibody of the present invention as described above conjugated directlyor indirectly to a detectable label. The antibody can be conjugateddirectly to the detectable label so that a covalent link exists betweenthe label and the antibody. Such methods are well known in the art; suchtechniques are described, for example, in G. T. Hermanson, “BioconjugateTechniques” (Academic Press, San Diego, 1996), incorporated herein bythis reference. In general, such techniques link reactive groups on theantibody and the label, typically through a linker or spacer. The lengthof the linker or spacer can be adjusted to preserve the activity andspecificity of the antibody and to ensure that the label produces thedetectable signal without interfering with the activity of the antibody.

Such labels are well known in the art and need not be described indetail. Such labels can include, but are not limited to, an enzymelabel, a radioactive label, a fluorescent label, a chemiluminescentlabel, a bioluminescent label, or a particulate label.

Enzyme labels are well known in the art. Typically, the enzyme used isone that produces a visually detectable signal such as a colored productor an insoluble product. Among the enzymes that can be used arehorseradish peroxidase, β-galactosidase, alkaline phosphatase, glucoseoxidase, urease, catalase, lysozyme, malate dehydrogenase,glucose-6-phosphate dehydrogenase, and ribonuclease A. Other enzymesthat can be used for immunoassays are well known in the art.

Among the particulate labels that can be used are latex labels andcolloidal metal labels such as colloidal gold, silver, tin, and othermetals.

A large variety of radioactive, fluorescent, chemiluminescent, andbioluminescent labels are known in the art. These labels need not bedescribed further here.

As an alternative to a direct covalent linkage between the antibody andthe label, the linkage can be indirect such as through a biotin-avidinlinkage. The binding of biotin to avidin or its bacterial analogstreptavidin is well understood in the art. This binding is highlyspecific and has an extremely high affinity. Typically, the antibody isconjugated to a biotin moiety and the label is bound to avidin orstreptavidin. Other arrangements can also be used. The use of anavidin-biotin linkage is described, for example, in G. T. Hermanson,supra, pp. 570-592.

III. Immunoassays

Another aspect of the present invention is immunoassays for tacrolimususing the antibodies described above. In general, such an immunoassay isa method of detecting or determining tacrolimus. The term “detecting” isused herein to refer to a qualitative assay that detects the presence orabsence of tacrolimus in a sample, while the term “determining” refersto a quantitative or semiquantitative assay that determines theconcentration of tacrolimus in the sample.

In general, such an immunoassay method comprises the steps of:

(1) providing a sample suspected of containing tacrolimus;

(2) reacting the sample with:

-   -   (a) an antibody against tacrolimus as described above; and    -   (b) optionally, with a tacrolimus analogue; wherein one of the        antibody or the tacrolimus analog is labeled with a label        producing a detectable signal; and

(3) observing or measuring one of:

-   -   (a) a signal associated with tacrolimus bound to antibody;    -   (b) a signal associated with tacrolimus unbound to antibody; or    -   (c) total signal present to detect or determine the presence or        concentration of tacrolimus in the sample.

Such assays are described as either homogeneous or heterogeneous. In aheterogeneous assay, the antibody bound to antigen is separated fromantibody unbound to antigen. This separation can be done by a number ofsteps well known in the art, such as differential solubility, reactivitywith another antibody, or other properties. Such assays are well knownin the art and need not be described further in detail here. If eitherthe signal associated with tacrolimus bound to antibody or the signalassociated with tacrolimus unbound to antibody is to be detected ordetermined, a heterogeneous assay is performed. By contrast, in ahomogeneous assay, the total signal present is detected or determined.In a homogeneous assay, the existence of an antigen-antibody complexmodulates the signal so that the signal level changes without arequirement of separating antigen bound to antibody from antigen unboundto antibody.

Although many heterogeneous immunoassay formats are well known in theart, in the context of the present invention, it is generally preferredto use a homogeneous immunoassay system. An example of a preferredhomogeneous immunoassay system for the immunoassay of tacrolimus usingantibodies according to the present invention is the homogeneous assaysystem known as EMIT, as described in D. D. Schottelius, “HomogeneousImmunoassay System (EMIT) for Quantitation of Antiepileptic Drugs inBiological Fluids” in Antiepileptic Drugs: Quantitative Analysis andInterpretation (C. E. Pippenger et al., Raven Press, New York, 1978, Ch.10, pp: 98-101, incorporated herein by this reference, and furtherdescribed in U.S. Pat. No. 3,817,837 to Rubenstein et al., incorporatedherein by this reference.

In general, in this assay, an enzyme such as glucose-6-phosphatedehydrogenase is conjugated to the analyte (here, tacrolimus) to beassayed in such a fashion that it does not significantly alter theactivity of the enzyme. However, when this analyte-enzyme conjugate isbound to the antibody for the analyte, the configuration is such thatthe active site of the enzyme is blocked and thus excludes thesubstrate. This results in the enzyme activity being reduced. When thecomplex is not bound, the active site is available to interact with thesubstrate. If free analyte is present in the unknown sample, the freeanalyte then binds to the antibody. This prevents the binding of theanalyte-enzyme conjugate and decreases the antibody-induced inactivation of the enzyme activity in proportion to the concentration ofanalyte in the sample. Thus, in such a homogeneous assay, the greaterthe concentration analyte in the sample, the greater the signal producedby the enzyme label. This is a homogeneous assay as described above.

IV. Test Kits

Another aspect of the present invention is a test kit, particularly foruse in the EMIT homogeneous immunoassay described above.

Such a test kit comprises, packaged in separate containers:

(1) an antibody as described above; and

(2) a tacrolimus analogue labeled directly or indirectly with an enzymelabel.

The tacrolimus analogue can be labeled either directly or indirectly; itis generally preferred that the tacrolimus analogue is labeledindirectly with a biotinylated tacrolimus molecule and astreptavidinylated enzyme. However, the tacrolimus can be coupled atcarbon 22 with an enzyme suitable for use in the EMIT assay as describedabove.

Test kits can also include, in separately packaged containers, otheringredients such as substrates, buffers, stabilizers, coenzymes, andother ingredients required for the performance of the immunoassay.

The invention is illustrated by the following Examples. The Examples arefor illustrative purposes only and are not intended to limit theinvention.

EXAMPLES Example 1 Preparation of Carbon-22-Substituted Derivatives ofTacrolimus

To determine the feasibility of derivatizing tacrolimus at carbon 22, asa model for this molecule, derivatization of the closely relatedmolecule FK-520 was performed. FK-520 has the same structure astacrolimus except that is has an ethyl instead of an allyl group atcarbon 21.

For these experiments, reagents and solvents were commercial grades andwere used as supplied without further purification. The reaction withFK-520 on a small scale (0.9 mg) was performed as follows: To a solutionof FK-520 (0.9 mg, 1.1 μmol) in 0.23 mL of methanol was added sodiumacetate (1.5 mg, 18.3 μmol, 16 equiv.) and carboxymethoxylaminehydrochloride (3.0 mg, 13.7 μmol, 12 equiv.). The reaction was stirredat room temperature for 3 hours under argon. Solvent was then evaporatedby purging with a stream of argon. Thin layer chromatography in5:6:1.1:0.5% (CH₂Cl₂/EtOAc/MeOH/HOAc) on plates from Analtech (scored10×20 cm, 250 microns) of the residue afforded 2 geometric isomers ofFK-520 mono-oximes, enough to be analyzed by fast atom bombardment massspectroscopy. LSIMS of C₄₅H₇₂N₂O₁₄ would be expected to give a [M−H]⁻ of863.3. The relevant portions of the mass spectroscopy results are shownin FIGS. 1 and 2. FIG. 2 is an portion of the spectrum shown in FIG. 1expanded around the central peak.

Subsequently an analogous procedure was used to prepare the monooxime oftacrolimus. Tacrolimus was provided as a solid mixture in a capsulecontaining 1.02 mg of tacrolimus monohydrate and approximately a 6-foldexcess of sodium dodecyl sulfate (SDS). The solid mixture from 30capsules was combined and extracted 4 times with 12 ml each time ofethyl acetate. The combined organic extracts were concentrated under avacuum to give 49 mg of crude tacrolimus which was used directly in thefollowing reaction.

To the 49 mg of crude tacrolimus (theoretically 36.5 μmol) in 3.5 ml ofmethanol was added sodium acetate (16.7 mg, 204 μmol, 5.6 equiv.), andcarboxymethoxylamine hydrochloride (40 mg, 183 μmol, 5 equiv.). Thereaction was stirred at room temperature for 12 hours under argon.Solvent and volatile materials were removed at reduced pressure to givecrude solid residue. Preparative thin layer chromatography on pre-coatedsilica gel plates from Analtech (20×20 cm, 1000 microns) with30:70:11:0.6 (CH₂Cl₂/EtOAc/MeOH/HOAc) of the crude product affordedgeometric isomers of tacrolimus monooximes at white solid. LSIMS onC₄₆H₇₂N₂O₁₄ was expected to yield 899.4 as [M+Na]⁺ and 915.4 as [N+K]⁺.Results are shown in FIGS. 3 and 4. FIG. 4 represents the data of FIG. 3at higher resolution centered around the relevant portion of thespectrum.

¹H NMR at 250 MHz in CDCl₃ of the product of the reaction ofcarboxymethoxylamine hydrochloride with tacrolimus is shown in FIG. 5.¹³C NMR at 250 MHz in CDCl₃ of the product of the reaction ofcarboxymethoxylamine hydrochloride with tacrolimus is shown in FIG. 6.¹³CNMR at 500 MHz in CDCl₃ shown in FIG. 7 was that of tacrolimus as areference (i.e. underivatized tacrolimus). All NMR spectra were recordedon Bruker instruments.

Example 2 Preparation of Tacrolimus-Keyhole Limpet Hemocyanin Conjugate

To a solution of tacrolimus monooxime (32.3 mg, 36.8 μmol) in 1.05 mL ofanhydrous dimethylformamide was addedl-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) (11mg, 57.4 μmol, 1.5 equiv.) and N-hydroxysuccinimide (7.3 mg, 63.4 μmol,1.7 equiv.). The reaction was stirred at room temperature for 1 hourunder argon. The mixture was then added dropwise via a syringe to asolution of keyhole limpet hemocyanin (74 mg, 54% pure) in 5.0 mL ofphosphate buffered saline (0.1 M, pH 8.0) and 0.25 mL ofdimethylformamide. After stirring at room temperature for 2 hours, theresulting suspension was dialyzed (1×4 L, 4° C., 2 h) against PBS (10mM, pH 7.0).

The resulting mixture was then extracted 3× with CH₂Cl₂ to remove anytrace amount of unreacted tacrolimus monooximes. Quantitative analysisof the mixture was conducted using bicinchoninic acid (BCA) proteinassay solution to give 50 mg of immunogen in 8 ml of PBS (10 mM, pH7.0).

Determination of the hapten number using the TNBS method (A. F. S. A.Habeeb, Anal. Biochem. 14:328 (1966)) gave a hapten number of 1300. Theimmunogen was immediately frozen using a dry ice-acetone bath and keptat −20° C. for storage.

Example 3 Preparation of Tacrolimus-Biotin Conjugate

To a solution of tacrolimus monooxime (12 mg, 13.7 μmol) in 0.3 mL ofanhydrous dimethylformamide was added EDAC (4 mg, 20.9 μmol, 1.5 equiv.)and N-hydroxysuccinimide (2.7 mg, 23.4 μmol, 1.7 equiv.). The reactionwas stirred at room temperature for 1 h under argon. To this was addedtriethylamine (10 μL, 75 μmol) and a solution of LC-biotin (10 mg, 25μmol, 2 equiv.) in 1.0 mL of DMF. Stirring was continued for another 3 hand the mixture was concentration under high vacuum to give a colorlessresidue. Reversed phase preparative thin-layer chromatography (PTLC) ofthe crude product on a C-18 plate from Whatman (PKLC₁₈F, 20×20 cm, 1000microns) (13:7 MeOH/H₂O) afforded 8 mg of the product as a white solid.The expected result from LSIMS of C₆₄H₁₀₃N₇O₁₆S is [M+H]⁺ of 1258.5. Themass spectroscopy results are shown in FIGS. 8 and 9. FIG. 9 representsa section of the spectrum of FIG. 8 at higher resolution centered aroundthe region of interest. ¹H NMR at 350 MHz in CDCl₃ was performed; theresults are shown in FIG. 10.

This is an example of derivatization of tacrolimus at position 22.

Example 4 Preparation of Bromoacetyl Derivative of Tacrolimus

Another derivative of tacrolimus derivatized at position 22 is abromoacetyl derivative. The bromoacetyl derivative can react asulfhydryl residue of an enzyme or another protein to produce atacrolimus-enzyme conjugate.

To form the bromoacetyl derivative of tacrolimus, first, to a solutionof succinimidyl bromoacetate (740 mg, 3.14 mmol) in 6 ml oftetrahydrofuran (THF) at 4° C. under argon was added, dropwise via asyringe, neat mono-N-BOC-ethylenediamine. After the addition wascomplete, the reaction was warmed to room temperature and stirred for 3h. The reaction solution was then concentrated in vacuo, and the residuedissolved in ethyl acetate. The ethyl acetate layer was washed with H₂Oonce, saturated aqueous NaHCO₃ twice, and brine once, dried over MgSO₄,and evaporated to dryness to give 440 mg of crude white solid.Purification on silica gel using Chromatotron (1:19 MeOH/CH₂Cl₂)afforded 386 mg of pure bromoacetyl ethylenediamine-mono-t-BOC as awhite solid.

To a solution of bromoacetyl ethylenediamine-mono-t-BOC (50 mg, 0.178mmol) in 2 ml of CH₂Cl₂ at room temperature was added dropwise via asyringe neat trifluoroacetic acid (TFA) (0.29 ml, 3.76 mmol). Thereaction was stirred for 3 h and then concentrated under vacuum. Traceamounts of TFA were azeotropically removed with toluene, and theresulting yellow oily product, the TFA salt of bromoacetylethylenediamine, was used directly in the next reaction.

To a solution of the carboxymethyloxime derivative of tacrolimus(derivatized at position 22) (100 mg, 0.14 mmol) andN-hydroxysuccinimide (NHS) (19 mg, 0.165 mmol) in 2 ml of THF underargon was added via a syringe neat diisopropyl carbodiimide (DIC) (23μl, 0.147 mmol). The reaction was stirred at room temperature for 3 hand was then transferred via a syringe to a solution of the TFA salt ofbromoacetyl ethylenediamine (0.178 mmol) in 3 mL of THF. To thisreaction solution was then added neat diisopropylethylamine (DIEA) (40μl), 0.23 mmol). Stirring was continued for 2.5 h, and the reaction wasconcentrated in vacuo to give a crude oil. Purification on preparativethin-layer chromatography (TLC) (7:3:1 EtOAc/CH₂Cl₂/MeOH) afforded 44 mg(37%) of bromoacetyl tacrolimus as a colorless solid.

Example 5 Preparation of Monoclonal Antibody to Tacrolimus

Preparation of monoclonal antibody to tacrolimus was performed asfollows. The immunogen was the tacrolimus conjugate with keyhole limpethemocyanin of Example 2. This immunogen was used to immunize Balb/cmice. The first immunization was 25 μg in a volume of 200 μl withmonophosphoryl lipid A and synthetic trehalose dicorynomycolate adjuvant(RIBI MPL+TDM Emulsion, RIBI ImmunoChem Research Inc.)intraperitoneally. Five weeks later a boost immunization was given with25 μg of the immunogen in 200 μl of monophosphoryl lipid A and synthetictrehalose dicorynomycolate adjuvant intraperitoneally. Subsequently,after another 8 weeks, a prefusion boost was given of the 25 μg of theimmunogen in 200 μl of Hanks' Balanced Salt Solution intravenously andintraperitoneally.

Three days later the fusion was performed by standard methods using anonsecreting murine myeloma designated P3×63-AG8.653.

Cloning was done by standard methods.

The clones were screened by the following reverse ELISA immunoassayprocedure according to the following protocol. Plates were coated withpolyclonal goat anti-mouse IgG (IgG+IgA+IgM) (Zymed) at 5 μg/ml inphosphate buffered saline at 100 μl per well. Plate coating wasperformed for 2 hours or more at room temperature or overnight at about4° C.; the plates could be stored wrapped in film at about 4° C. forseveral days. The plates were then flicked dry and blocked with 300 μlper well of blocking buffer diluent (0.5% bovine serum albumin, 0.05%Tween 20 in PBS). Plate blocking was performed by incubation for 15minutes or more at room temperature with plate shaking. The plates werethen flicked dry. The monoclonal antibody to be screened was then addedto each well as follows: 50 μl per well of blocking buffer diluent wasadded along with 50 μl per well culture supernatant transferred from thecorresponding well in the fusion growth plate. Incubation was for about1 hour at room temperature with plates shaking. The plate was washedusing a Titerteck Plus plate washer with S20 stacker with the washingbuffer being PBS with 0.05% Tween 20. An enzyme conjugate of tacrolimuscovalently coupled to glucose-6-phosphate dehydrogenase diluted inblocking buffer diluent to 1:4000 was added at 100 μl per well.Incubation was performed for about 1 hour at room temperature withshaking. The plate was then washed and a chromogenic solution was addedat a volume of 100 μl per well. The chromogenic solution contained 0.593mM p-iodonitrotetrazolium violet, 0.02 M NAD, 0.033 Mglucose-6-phosphate, 0.055 M Tris, 0.02% sodium azide, and a 1:4000dilution of diaphorase (lipoyl dehydrogenase) (Sigma, St. Louis, Mo.).BSA was present at 1% (vol/vol) of a 5% w/vol BSA solution. BSA was usedto help prevent rapid precipitation of reduced p-iodonitrotetrazoliumviolet.

From the screening a hybridoma producing a suitable monoclonal antibodywas selected. This is designated as 1H6 and is a IgG₁λ antibody. Thisantibody has a binding affinity for tacrolimus of about 3.7×10⁹liters/mole, that cross-reacts with 13-demethyl tacrolimus, and that hasless than about 8% cross-reactivity to all of the following tacrolimusmetabolites: 15-demethyl tacrolimus; 31-demethyl tacrolimus;13,31-didemethyl tacrolimus; 15,31-didemethyl tacrolimus; and 12-hydroxytacrolimus.

Example 6 Comparison of Cross-Reactivity with Tacrolimus Metabolites forMonoclonal Antibody of Example 4 and Other Antibodies

The antibody of Example 5, another antibody resulting from the cloningand designated 14H04, and other antibodies, were tested forcross-reactivity of tacrolimus metabolites (13-demethyl tacrolimus,15-demethyl tacrolimus, 31-demethyl tacrolimus, 13,31-didemethyltacrolimus, 15,31-didemethyl tacrolimus, and 12-hydroxy tacrolimus). Allof these metabolites except 12-hydroxy tacrolimus were tested at variouslevels in hemolysate containing 10 ng/ml of tacrolimus; 12-hydroxytacrolimus was tested at 10 ng/ml with no tacrolimus in the sample.Methanolic extracts were used in this testing. The standard curvegenerated for this testing were verified using MORE immunosuppressivedrug controls level 1-4, a standard control for these assays (MoreDiagnostics, Los Osos, Calif.).

All antibodies showed cross-reactivity to 13-demethyl tacrolimus tovarying degrees. Cross-reactivity was calculated by the followingformula: (cross-reaction value ng/ml)-solvent control value(ng/ml)×100)/metabolite spiked target (ng/ml)=% cross-reactivity.

The results are shown in Table 1. With the exception of 13-demethyltacrolimus, the 1H6 antibody of Example 5 had less than 8%cross-reactivity with the other analytes tested. TABLE 1CROSS-REACTIVITY OF ANTIBODIES WITH TACROLIMUS METABOLITES Metabolite inTacrolimus Hemolysate Present 14H04 1H6 6D5-D11 6D5-D12 6D5-F1213-demethyl 10 ng/mL 227%  78%  120%  87%  65% tacrolimus 5.0 ng/mL15-demethyl 10 ng/mL  8% 5%  9% 4% 0 tacrolimus 5.0 ng/mL 31-demethyl 10ng/mL 31% 7% 13% 8% 0 tacrolimus 5.0 ng/mL 13, 31 di 10 ng/mL 10% 0 42%0 0 demethyl tacrolimus 5.0 ng/mL 15, 31 di 10 ng/mL 0 0 29% 14%  0demethyl tacrolimus 5.0 ng/mL 12-OH tacrolimus  0 ng/mL 0 0 0 0 0  10ng/mL

Example 7 Homogeneous Immunoassay Using Monoclonal Antibody toTacrolimus

A homogeneous immunoassay to tacrolimus based on the EMIT procedure wascarried out according to the following protocol. A volume (200 μl) ofsample or calibration standard was pipetted into a microfuge tube. Intothe same tube, 50 μl of 300 mM CuSO₄ was pipetted along with 200 μl ofmethanol. The mixture was capped and vortexed for 10 seconds andcentrifuged for 5 minutes at 20800×g. The supernatant was decanted intoa sample cup of a Roche Cobas Mira analyzer according to standardparameters for that analyzer. The following reagents were placed intothe reagent rack of the analyzer: Reagent A contained NaCl, Na₂EDTA,anti-tacrolimus monoclonal antibody (1H6), nicotinamide adeninedinucleotide (NAD), glucose-6-phosphate, detergent (Pluronic 25R2),sodium azide, and n-methylisothiazolone at pH 5.5. Reagent B was Tris,pH 8.2, with Na₂EDTA, detergent (Pluronic 25R2), sodium azide, andn-methylisothiazolone. Reagent C was a tacrolimus-glucose-6-phosphatedehydrogenase conjugate at pH 7.0, in Na₂HPO4, Na₂EDTA, bovine serumalbumin, detergent (Pluronic 25R2), and sodium azide. The sample andreagent racks are loaded onto the analyzer and the run is startedaccording to the parameters that have been preset. The enzyme rates areprinted out during the run in terms of milli-od/min. The calibrationcurves are shown in FIG. 11 for 1H6 and another monoclonal antibodydesignated as 14H04.

Example 8 Correlation Between Assay for Tacrolimus Using MonoclonalAntibody of the Present Invention and EMIT Assay with LC-MS/MS Method

A series of samples from 70 patients to whom tacrolimus had beenadministered was assayed with the EMIT-homogeneous immunoassay using the1H6 monoclonal antibody of the present invention as compared with theLC/MS/MS method. The LC/MS/MS method is an assay method for tacrolimusthat employs liquid chromatography followed by tandem mass spectroscopy(P. J. Taylor et al., “Sensitive, Specific Quantitative Analysis ofTacrolimus (FK506) in Blood by Liquid Chromatography-Electrospray TandemMass Spectrometry,” Clin. Chem. 42: 279-285 (1996)). The results areshown in FIG. 12. The results show a very high correlation with acorrelation coefficient of 0.929.

Example 9 Comparison of Results Using EMIT Homogeneous Immunoassay andLC/MS/MS Using Specimens from Patients with Liver Dysfunction

Although monoclonal antibody 1H6 has substantially minimizedcross-reactivity to metabolites of tacrolimus other than 13-demethyltacrolimus, some cross-reactivity remains with 13-demethyl tacrolimus.In order to determine whether or not the cross-reactivity with13-demethyl tacrolimus would interfere with use of this antibody in animmunoassay, a panel of specimens from 70 patients who had severe liverdysfunction and were awaiting transplantation while on tacrolimus wasassayed with the EMIT immunoassay using the 1H6 monoclonal antibody,with the LC/MS/MS method, and with another commercially availableimmunoassay for tacrolimus, the IMx immunoassay produced by Abbott. Suchsamples typically contain higher levels of tacrolimus metabolites thando those from patients who have undergone organ transplantation but havenormal liver function. Tacrolimus levels were measured on the panel ofsamples using these three assays.

The results are shown in FIG. 13 for the comparison between the EMITassay and the LC/MS/MS assay and in FIG. 14 for the comparison betweenthe EMIT assay and the IMx assay (Abbott). Deming's regression analysiswas used to interpret the results; the results are shown in Table 2. TheEMIT assay using the 1H6 monoclonal antibody had closer agreement withLC/MS/MS than the Abbott IMx assay based on the slope of the results(see Table 2) with the slope of 1.11 for EMIT and 1.45 for IMx. Averageconcentration of the 70 samples of 6.79 ng/ml by MC/MS/MS, 6.96 ng byEMIT using the 1H6 monoclonal antibody, and 7.93 ng/ml by Abbott IMx.Two samples tested above range by IMx only. Bland-Altman analysis of the70 results are shown in FIG. 15 for EMIT using monoclonal antibody 1H6and Abbott IMx versus LC/MS/MS. These plots illustrate that in generalnoticeable variability of results versus LC/MS/MS occurs with samplesabout 10 ng/ml. The pattern of difference for LC/MS/MS by immunoassaywas similar for the EMIT assay using monoclonal antibody 1H6 and theAbbott IMx assay. TABLE 2 Correlation Between Results of Assays forTacrolimus in Patients with Liver Damage Comparison Slope/InterceptCorrelation EMIT vs. LC/MS/MS y = 1.11x − 0.66 0.910 IMx vs. LC/MS/MS y= 1.47x − 2.11 0.925 EMIT vs. IMx y = 0.77x + 0.87 0.953

Even in these patients containing high levels of tacrolimus metabolites,the assay using the monoclonal antibody 1H6 in an EMIT homogeneousimmunoassay showed a high degree of correlation relative to othermethods. Therefore, liver dysfunction may not cause a severe impact onthis assay using this antibody and the results imply that clinicalmanagement of patients by immunoassay can be accomplished similarly byusing the EMIT immunoassay with the 1H6 monoclonal antibody or thepreviously-available Abbott IMx immunoassay for tacrolimus. This showsthe clinical capability of the 1H6 monoclonal antibody.

Advantages of the Present Invention

The present invention provides a monoclonal antibody to tacrolimus thatminimizes cross-reactivity with tacrolimus metabolites. The monoclonalantibody of the present invention can be used in an improved immunoassayfor tacrolimus that correlates well with other immunoassay methods andnon-immunological assay methods for tacrolimus. An immunoassay employinga monoclonal antibody is suitable for clinical monitoring of patientsreceiving tacrolimus, including patients with impaired liver functionwho are expected to have high levels of tacrolimus metabolites in theirserum.

Although the present invention has been described with considerabledetail, with reference to certain preferred versions thereof, otherversions are possible. Therefore, the spirit and scope of the appendedclaims should not be limited to the description of the preferredversions contained herein.

1. (canceled)
 2. A monoclonal antibody to tacrolimus that: (a) competeswith the IgG₁λ monoclonal antibody designated 1H6 at least about 80% aseffectively on a molar basis as compared with the IgG₁λ monoclonalantibody designated 1H6 as measured by competition assays; and (b) hasless than about 10% cross-reactivity with each of 15-demethyltacrolimus, 31-demethyl tacrolimus, 13,31-didemethyl tacrolimus,15,31-didemethyl tacrolimus, and 12-hydroxy tacrolimus.
 3. Themonoclonal antibody of claim 2 wherein the antibody competes at leastabout 90% as effectively on a molar basis as the monoclonal antibodydesignated 1H6 and has less than about 8% cross-reactivity with each of15-demethyl tacrolimus, 31-demethyl tacrolimus, 13,31-didemethyltacrolimus, 15,31-didemethyl tacrolimus and 12-hydroxy tacrolimus. 4.(canceled)
 5. A hybridoma producing the monoclonal antibody of claim 2.6. The monoclonal antibody of claim 2 wherein at least some of theconstant regions of the antibody are replaced by human constant regionsso that the monoclonal antibody is humanized.
 7. The monoclonal antibodyof claim 6 wherein the antibody competes at least about 90% aseffectively on a molar basis as compared with the IgG₁λ monoclonalantibody designated 1H6 and wherein the antibody has less than about 8%cross-reactivity with each of 15-demethyl tacrolimus, 31-demethyltacrolimus, 13,31-didemethyl tacrolimus, 15,31-didemethyl tacrolimus,and 12-hydroxy tacrolimus. 8-18. (canceled)
 19. A conjugate comprisingthe antibody of claim 2 conjugated directly or indirectly to adetectable label.
 20. The conjugate of claim 19 wherein the detectablelabel is selected from the group consisting of an enzyme label, aradioactive label, a fluorescent label, a chemiluminescent label, abioluminescent label, and a particulate label.
 21. The conjugate ofclaim 20 wherein the label is an enzyme label.
 22. A conjugatecomprising the antibody of claim 6 conjugated directly or indirectly toa detectable label.
 23. The conjugate of claim 22 wherein the detectablelabel is selected from the group consisting of an enzyme label, aradioactive label, a fluorescent label, a chemiluminescent label, abioluminescent label, and a particulate label.
 24. The conjugate ofclaim 23 wherein the label is an enzyme label. 25-41. (canceled)
 42. Amethod of detecting or determining tacrolimus comprising the steps of:(a) providing a sample suspected of containing tacrolimus; (b) reactingthe sample with: (i) the antibody of claim 2; and (ii) optionally, atacrolimus analogue; wherein one of the antibody or the tacrolimusanalogue is labeled with a label producing a detectable signal; and (c)observing or measuring one of: (i) the signal associated with tacrolimusbound to antibody; (ii) the signal associated with tacrolimus unbound toantibody; or (iii) the total signal present; in order to detect ordetermine the presence or concentration of tacrolimus in the sample. 43.The method of claim 42 wherein the sample is reacted with a tacrolimusanalogue labeled with an enzyme label and the total signal present isobserved or measured to detect or determine the presence orconcentration of tacrolimus in the sample. 44-56. (canceled)
 57. A testkit comprising, packaged in separate containers: (a) the antibody ofclaim 2; and (b) a tacrolimus analogue labeled directly or indirectlywith an enzyme label. 58-82. (canceled)